Mutations in DONSON disrupt replication fork stability and ... · PDF fileRS4819274 48Mb BB BB...

Supplementary Information

Mutations in DONSON disrupt replication fork stability and cause

microcephalic dwarfism.

Nature Genetics: doi:10.1038/ng.3790

DONSON mutation: S28R

HUMAN 1-MALSVPGYSPGFRKPPEVVRLRRKRARSRGAAASPPRELTEPAARRAALVAGLPLRPFPA 60

CHIMP 1-MALSVPGYSPGFRKPPEVVRLRRKRARSRGAAASPPRELTEPAARRAALVAGLPLRPFPA 60

PIG 1-MALSVPGYSPSFKRPPETLRLRRKRGRSLGAAP-PPKERPEQTTRRAALAAGLPLRPFPA 59

COW 1-MDLSVPGYSPSFKRPPETLRLRRKRGRSLGAA-----ERPEPATRRAARAAGLPLRPFPA 55

DOG 1-MAVSVPGYSPGFRKPPATLRLRRKRARSHGSAA-APGEQPEPAPRRAALAAGLLLRPFPA 59

MOUSE 1-MAVSVPGYSPSFKRPPETVRLRRKRSRDHGAAV--PASLPEPAPRRAALAAGLPLRPFPT 58

DONSON mutations: C278R, Y282C, F292L

HUMAN 235-IGADRKMAGKTSPWSNDATLQHVLMSDWSVSFTSLYNLLKTKLCPYFYVCTYQFTVLFRA 294

CHIMP 235-IGADRKMAGKTSPWSNDATLQHVLMSDWSVSFTSLYNLLKTKLCPYFYVCTYQFTVLFRA 294

PIG 231-IGADRKMAGKANPWSNDETLQHNLLSDWSVSLTSLYNLLKTKLCPYFYVCTYQFTILFRA 290

COW 227-IGADRKMAGKTTPWSNDETLQHVLMSDWSVSFTSLYNLLKTKLCPYFYVCTYQFTILFRA 286

DOG 240-IGADRKMAGKISPWSNDETLQHILMSDWSVSFTSLYNLLKTKLCPYFYVCTYQFTILFRA 299

MOUSE 239-IGADRKMAAKTSPWSADETLQHALMSDWSVSFTSLYNLLKTKLCPYFYVCSYQFTVLFRA 289

DONSON mutation: M446T

HUMAN 421-LVATSGPQAGLPPTLLSPVAFRGATMQMLKARSVNVKTQALSGYRDQFSLEITGPIMPHS-480

CHIMP 421-LVATSGPQAGLPPTLLSPVAFRGATMQMLKARSVNVKTQALSGYRDQFSLEITGPIMPHS-480

PIG 417-LVASSGPQAGLPPTLLSPIAFRGATMQMLKARSVNVKTQALSGYKDQFSLEITGPVMPHS-476

COW 413-LIATSGPQAGLPPTLLSPVAFRGATMQMLKARSVNVKTQALSGYRNQFSLEITGPIMPHS-472

DOG 426-LIASSGPQAGLPPTLLSPVAFRGATMQMLKARSVNVKTQAVSGYKDQFSLEITGPIMPHS-485

MOUSE 415-LVATSGAQAGLPPTLLSPIAFRGASMQMLKARSSNVKTQALSGYRDKFSLDITGPVMPHA-474

DONSON mutations: ΔN417-S418, P433S

HUMAN 375-KKPDILSIKLRKEKHEVQMDHRPESVVLVKGINTFTLLNFLINSKSLVATSGPQAGLPPT-434

CHIMP 375-KKPDILSIKLRKEKHEVQMDHRPESVVLVKGINTFTLLNFLINSKSLVATSGPQAGLPPT-434

PIG 371-KKPDVLSIKLRKEKHEVQMDHRPESVVLVKGMNTFTLLNFLINCKSLVASSGPQAGLPPT-430 COW 367-KKPDVLSIKLRKEIHEVQMDHRPESVVLVKGMNTFTLLNFLINCKSLIATSGPQAGLPPT-426

DOG 500-KKPDILSIKLRKEKHEVQMDHRPESVVLVKGMNTLTLLNFLINSKSLIASSGPQAGLPPT-551

MOUSE 369-KKPDVISIKLRKEKHEVQMDHRPESVVLVKGLNTFKLLNFLINCKSLVATSGAQAGLPPT-428

DONSON mutation: K489T, E504K

HUMAN 468-FSLEITGPIMPHSLHSLTMLLKSSQSGSFSAVLYPHEPTAVFNICLQMDKVLDMEVVHKE-527

CHIMP 468-FSLEITGPIMPHSLHSLTMLLKSSQSGSFSAVLYPHEPTAVFNICLQMDKVLDMEVVHKE-527

PIG 464-FSLEITGPVMPHSLHSVTMLLKSSQSGSFSAGLYTHEPTAVFNTCLPTDKVLDREAVLEE-523

COW 460-FSLEITGPIMPHSLHSVTMLLQSSQNGSFSAGLYTHEPTAVFNICPPKDNVLDKEAVHEE-519

DOG 593-FSLEITGPIMPHSLHSVTMLLRSSQNGSFSAGLYTHEPTAVFNICLPVNKVLDKETVLEE-652

MOUSE 462-FSLDITGPVMPHALHSMSMLLRSSQRGSFSAGLYAHEPTAVFNVGLSLDKELDRKVARED-511

Supplementary Figure 1: Conservation of DONSON amino acids mutated in MD patients.

Amino acid alignment of DONSON protein from different species showing the degree of evolutionary

conservation of disease causing DONSON point mutations, generated using Clustal Omega. Red

arrows indicate the amino acid residues of DONSON mutated in MD patients. The K489T and S28R

missense variants associated with the haplotype present in patients P1-7 are coloured red. All other

mutations are coloured blue.


Supplementary Figure 2: Cerebral cortical size is markedly reduced with simplification of

gyral folding in patients with DONSON mutations.

Magnetic resonance imaging T1-weighted sagittal and axial FLAIR images of patient P5, and T2-

weighted sagittal and axial images of patients P6 and P7, compared with age-matched control

scans of healthy individuals. Scale bars, 10 cm.


Supplementary Figure 3: Clinical and genetic characterisation of affected individuals from

family P21.

(a) and (b) are clinical photographs of dead fetuses (P21-1 and P21-2). (c) Pedigree of family P21.

(d) IBDelphi image of chromosome 21. (e) Chromatograms indicating the presence of the c.1047-

9A>G mutation within both affected fetuses, and a parent, from family P21.

DONSON

(chr21:34,949,859-34,959,908) c.1047-9A>G

Contr

ol

Pare

nt

Aff

ecte

d

a b

c

d e

P21-1 P21-2

Aff

ecte

d


SNPs Distance Mb P14 P15 P16 P17 P18-1 P18-2 P18-3

RS7279441 24.1Mb AA AA AA ab AA AA AA

RS1118467 24.2Mb ab ab BB AA ab ab ab

1332 markers

RS171487 29Mb ab BB BB BB ab ab ab

RS2831134 29Mb AA AA AA AA ab ab ab

142 markers

RS2831813 29.8Mb BB BB ab BB BB BB BB

RS4420778 29.8Mb BB BB BB BB BB BB BB

519 markers

RS2255507 32.4Mb BB AA AA BB ab ab ab

RS2833282 32.4Mb AA AA AA AA AA AA AA

440 markers

RS17632819 34.4Mb BB ab BB AA BB BB BB


67 markers

RS17860260 34.6Mb BB BB BB BB AA AA AA



33 markers


DONSON 34.9Mb AA AA AA AA AA AA AA

DONSON 34.9Mb BB BB BB BB BB BB BB

RS2070392 35Mb BB BB BB BB BB BB BB

280 markers



RS2298352 36.2Mb BB BB AA BB BB BB BB

280 markers


RS11910121 37.3Mb ab BB BB BB BB BB BB

326 markers

RS2211845 39.1Mb AA AA AA BB BB BB BB

RS8133218 39.1Mb BB BB BB BB ab ab ab

1253 markers


RS150796 43.4Mb ab AA ab BB BB BB BB

436 markers

RS4819274 48Mb BB BB BB AA BB ab BB

RS9637 231 48Mb AA AA ab BB AA ab AA

-

-

-

- - -

Schematic of high density SNP genotyping of the DONSON locus in P14, P15, P16, P17, P18-1, P18-2 and P18-3 (Color Shading). Heterozygous SNP markers delineating regions of homozygosity are shown in red. An identical haplotype of 321 SNPs is evident in the six patients within ~1.6Mb shared

region of homozygosity consistent with a shared common ancestor (Grey shading). 313 homozygous SNP markers within the 1.6 Mb regions are omitted for clarity.

Supplementary Figure 4: Common ancestral haplotype flanking the DONSON locus in

families P14 to P18-3.

Patients P14, P15, P16, P17, P18-1, P18-2 and P18-3 have a distant common ancestry, sharing a

common ~1.6 Mb homozygous haplotype across the DONSON locus. Schematic of high density

SNP genotyping of the DONSON locus in the six patients indicated above. The different families are

indicated by different shades of blue. Heterozygous SNP markers delineating regions of

homozygosity are shown in red. An identical haplotype of 321 SNPs is evident within the 1.6 Mb

region of homozygosity in the six patients, which is consistent with a shared common ancestor

(indicated by grey shading). 313 homozygous SNP markers within the 1.6 Mb regions are omitted

for clarity.


Exon4 Exon 5

Patient P18-1 (c.786-22A>G)

Exon4 Exon 6

a b

F R

Ex4 Ex5 Ex6 Ex7

500bp

400bp

300bp

200bp

100bp

P18-1 P18-2 WT

GA

PD

H

DO

NS

ON

0

0.2

0.4

0.6

0.8

1

1.2Mutant

Wild Type

Rela

tive

DO

NS

ON

tra

nscri

pt le

ve

ls

DONSON c.786-22A>G

WT DONSON

c

WT

Supplementary Figure 5: The patient-associated DONSON intronic mutation c.786-22A>G

affects mRNA splicing.

(a) Top: a schematic of DONSON mRNA with the position of the PCR primers (F = forward primer;

R = reverse primer) indicated relative to the exons. Bottom: PCR amplification of DONSON exons 4-

7 from cDNA derived from a heathy normal WT individual or patients 18-1 and 18-2. PCR

amplification of GAPDH was used as a control. (b) Sequencing electropherogram of the PCR

products highlighted in (a). The red box indicates the WT sequence. The green box indicates

skipping of exon 5 of DONSON. (c) Quantification of the relative amounts of the WT DONSON

mRNA as compared to the levels of GAPDH by RT-qPCR from three heathy normal individuals and

four DONSON patients (n = 3 independent experiments). P values were determined by a ’

t-test (c).

P = 3.2E-22


200 bp

300 bp

500 bp

800 bp c.7

86-3

3A

>G

(P

2)

c.7

86-3

3A

>G

(P

6)

c.7

86-2

2A

>G

(P

12)

c.7

86-1

G>

A

WT

Untr

ansfe

cte

d

5’ Ex4 Ex5 3’

F-primer

R-primer

RSV promoter

c.786-33A>G

c.786-22A>G

c.786-1G>A

Intron 3 Intron 4 Intron 5

Exon 4 Exon 5 5’ 3’

Exon 4 5’ 3’

5’ 3’

a

b


does not affect mRNA splicing.

(a) A schematic of DONSON mRNA splicing mini gene reporter construct with the relative positions

of the PCR primers, RSV promoter, two artificial vector-associated exons (labelled as 5’ and 3’ and

DONSON exons/introns indicated. The location of the two DONSON intronic variants (c.786-22A>G

and c.786-33A>G) relative to the intron/exon boundaries are shown. An essential splice site c.786-

1G>A mutation was generated as a positive control that disrupts splicing. The position of the PCR

primers (F-primer = forward primer; R-primer = reverse primer) is indicated relative to the exons. (b)

PCR amplification of DONSON exons 4-5 from cDNA derived from the splicing reporter construct

containing WT or the individual DONSON mutations. The exon content of each PCR product was

verified by sequencing (indicated).


WT

S2

8R

M4

46

T

K4

89

T

-DOX

WT

DONSON Con siRNA:

DONSON

CHK2

HeLa

Supplementary Figure 7: Depletion of endogenous DONSON in HeLa Flp-In/T-Rex

cells

Endogenous DONSON was depleted using siRNA from HeLa Flp-In/T-Rex cells

expressing doxycycline-inducible GFP-tagged siRNA-resistant WT or mutant DONSON.

Endogenous DONSON were detected using an anti-DONSON antibody. CHK2 was

used as a loading control.


Supplementary Figure 8: Alterations in the protein level of the K489T GFP-DONSON variant

are not due to reduced levels of mRNA

(a) Immunoblotting using an anti-GFP antibody demonstrates reduced protein levels of the K489T

GFP-DONSON variant compared to WT GFP-DONSON (same cell lines as Fig 2d). ‘+T ’

expression induced by treatment with 1 ug/ml tetracycline for 24 h; ‘-T ’ uninduced. α-Tubulin was

used as a loading control. (b) qRT-PCR analysis of GFP-DONSON transcript levels from RNA

isolated from the same cell pellets as in (a). Transcript levels are expressed relative to the

housekeeping gene PBGD. Data in (a) and (b) are representative of three technical replicates from

two independent experiments. P values were determined by a ’ t-test (b).

0

0.1

0.2

0.3

0.4

0.5

GF

P-D

ON

SO

N T

ranscrip

t re

lative e

xpre

ssio

n

K489T

WT

-Tet +Tet

K489T

WT

a b

α-Tubulin

GFP-Donson K

489T

WT

-Tet +Tet

K489T

WT

P = 0.53


N417_S418del S28R

GF

P

DA

PI

Merg

e

GFP

Vector K489T F292L Y282C Q543_I544insK M446T

WT

DONSON

a

b

0%

20%

40%

60%

80%

100%

Diffuse

Nuclear

Supplementary Figure 9: The patient-associated DONSON point mutations disrupt protein

sub-cellular localisation.

(a) Patient-associated point mutations in DONSON affect its subcellular localization. HeLa cells

were transfected with a vector encoding either WT GFP-DONSON, or GFP-DONSON containing

patient-derived mutations and fixed 24 h post-transfection. The percentages of GFP positive cells

that contained either a solely nuclear GFP signal, or a diffuse pan-cellular GFP signal were

quantified (n = 5 independent experiments). P values between WT and mutated GFP-DONSON

were determined by a ’ t-test. (b) Representative images are shown. Missense mutations

associated with the haplotype in patients P1 to P7 are shaded red and all other mutations are

shaded blue. Scale bars; 10 µm.

Perc

enta

ge o

f cells



affects mRNA abundance.

(a) Schematic presentation of exon and intron boundaries of a region of DONSON. Arrows indicates

primers (F; forward: and R; reverse) used for semi-quantitative RT-PCR and qRT-PCR. (b) Semi-

quantitative RT-PCR analysis of DONSON transcript levels in P21-1 and P21-2 and three normal

controls (NC-1, NC-2 and NC-3). Red star indicates retention of intronic region in patient amplicon.

(c) qRT-PCR analysis of DONSON transcript levels, normalised to GAPDH, in patients P21-1, P21-

2 and three healthy normal controls (NC) (n = 3 independent experiments). Error bars indicate s.d.

(a.u. = arbitary units).

a

b

F: CAACTCGAGGTTTAAGAGAAGC R: CATCCTCATCACTGATGGC

P21-2 P21-1 NC-1 NC-2 NC-3 P21-2 P21-1 NC-1 NC-2 NC-3

DONSON GAPDH

*

100 bp

200 bp 300 bp

109 bp

Exon-5 Exon-6 Exon-7

-9A>G

F R

c

NC P21-1 P21-2

DONSON transcript expression 1.2

1.0

0.8

0.6

0

Rela

tive e

xpre

ssio

n (

a.u

.)

0.4

0.2


H2A

DONSON

1 mM HU (h)

WT1

0 8 24

P12

0 8 24

P2

0 8 24

P9

0 8 24

WT2

0 8 24

P6

0 8 24

P10-2

0 8 24

H2A

DONSON

1 mM HU (h)

Supplementary Figure 11: Patient derived fibroblasts retain residual levels of DONSON

protein.

Immunoblotting of cell extracts from patient derived fibroblast cell lines treated with HU for the

indicated times. WT1 and WT2 indicate unrelated healthy normal individuals. H2A was used as a

loading control.


0

10

20

30

40

50

60

70

80

90

EdU +ve EdU -ve

Perc

enta

ge o

f cells

with >

10 5

3B

P1 foci

Control DONSON

DONSON

γ-H2AX

H2A

siRNA:

a

d

DO

NS

ON

siR

NA

C

ontr

ol siR

NA

53BP1

DAPI 53BP1

DAPI EdU

EdU

b

Supplementary Figure 12: Depletion of DONSON gives rise to increased spontaneous DNA

damage in replicating cells.

(a) Knockdown of DONSON results in increased levels of phosphorylated H2AX. HeLa cells were

transfected with control or DONSON siRNA, harvested 72 h post-transfection and extracts subjected to

SDS-PAGE and immunoblotting using the antibodies indicated (n = 3 independent experiments). (b-e)

DONSON depletion causes DNA double strand breaks in replicating cells. (b) DONSON-depleted cells

were seeded onto coverslips and incubated with 10 μ EdU for 10 min before harvesting. Cells were

then pre-extracted, fixed and stained with an antibody to γ 2AX. EdU was visualised using the Click-iT-

Alexa-488 Imaging Kit. The intensity of γ 2AX immunostaining in >100 EdU positive cells and >50 EdU

negative cells were quantified per cell line per condition per experiment (n = 2 independent

experiments). (c) Quantification of the percentage of EdU positive and negative cells with >10 53BP1

foci in cells without DONSON. A minimum of 300 cells were counted per sample per experiment (n = 3

independent experiments). (d) Representative images for (c) Scale bars; 10 µm. (e) Pulsed-field gel

electrophoresis was used to detect the presence of DNA double strand breaks (DSBs) in cells from (a).

As a positive control, HeLa cells were irradiated with 30 Gy of ionising radiation (IR) and harvested 1

later. Throughout, P values were determined by a Mann–Whitney U test (b), or a ’ t-test (c).

Control

siRNA

DONSON

siRNA

Control

siRNA

DONSON

siRNA

EdU negative EdU positive

0

2x107

4x107

6x107

8x107

1x108

ɣ AX

c

Control

siRNA

DONSON

siRNA IR (30Gy)

DSBs

e

P = 0.0020

P < 0.001


Supplementary Figure 13: Fluorescence Cross-Correlation Spectroscopy of GFP-DONSON and RFP-

PCNA. See next page for legend.

P = 0.00014

P = 0.094


Time (h) after

250ng/ml MMC

NBS1 S343-P

Control

siRNA

CHK1 S345-P

SMC1-S966-P

0

H2A

γ-H2AX

24

NBS1

CHK1

SMC1

RPA2-S4/8-P

RPA2

0 24

DONSON

siRNA

DONSON

FANCD2

a b

Supplementary Figure 14: Reduced ATR-CHK1-dependent signalling and defective

activation of the G2/M checkpoint in DONSON-depleted cells following replication stress

treatment

(a) Cells lacking DONSON exhibit defective or delayed ATR auto-phosphorylation in response to

replication stress. HeLa cells transfected with either control or DONSON siRNA were exposed to

1 mM HU or 250 ng/ml MMC for 24 h, and subjected to immunoblotting using the indicated

antibodies (n = 2 independent experiments). (b) Cells lacking DONSON fail to efficiently activate

the ATR-dependent replication stress response following exposure to MMC. Whole cell extracts of

HeLa cells transfected with either control or DONSON siRNA were subjected to immunoblot

analysis using the indicated antibodies following treatment with 250 ng/ml MMC for 24 h. (c) The

percentage of mitotic cells in cells from (a) was determined by flow cytometry using antibodies to

phosphorylated histone H3-Ser10 as a marker of mitotic cells. Data represents the mean of three

independent experiments. P values were determined by a ’ t-test (c).

CHK1 S317-P

Control siRNA

H2A

γ-H2AX

_

CHK1

RPA2-S4/8-P

RPA2

HU MMC

DONSON

DONSON siRNA

_ HU MMC

ATR T1989-P

ATR

c

0

5

10

15

20

25

30

Perc

enta

ge m

itotic c

ells

com

pare

d to u

ntr

eate

d c

ontr

ol

Con siRNA

Donson siRNADONSON siRNA

Control siRNA

+ MMC

P = 1.06E-07


RP

A flu

ore

scence in

tensty

(A

U)

Control

siRNA

DONSON

siRNA

Control

siRNA

DONSON

siRNA

0

20

40

60

80

+HU

a

RP

A

DA

PI

Control

siRNA

DONSON

siRNA

Control

siRNA

DONSON

siRNA

+HU

Supplementary Figure 15: Increased levels of RPA-coated ssDNA in DONSON-depleted cells

following treatment with HU

Exposure of cells lacking DONSON to HU induces increased levels of RPA coated ssDNA. (a-b) HeLa cells

transfected with either control or DONSON siRNA were exposed to 1 mM HU for 24 h, immunostained with

antibodies specific to RPA2 and analysed by fluorescence microscopy. Fluorescence intensity per nucleus

was quantified using ImageJ. Lines denote mean values from two independent experiments. Representative

images are shown in (b). (c-d) Native BrdU foci formation in HeLa cells transfected with the indicated

siRNAs was analysed by fluorescence microscopy. The cells were treated as detailed in the schematic

(top), and immunostained with antibodies to BrdU. Foci formation was quantified using ImageJ (n = 3

independent experiments). (d) Representative images are shown. As a control for BrdU incorporation, cells

were treated with 2 M HCl to denature DNA prior to addition of the BrdU antibody. Scale bars; 10 µm. P

values were determined by a Mann-Whitney U test (c).

60

0

20

40

Num

ber

of

native B

rdU

foci per

cell

Control

siRNA

DONSON

siRNA

Control

siRNA

DONSON

siRNA

+HU

b

c 6 h

Harvest 2 mM HU 10 µm BrdU

24 h Control

siRNA

DONSON

siRNA

Control

siRNA

DONSON

siRNA

+HU

Merg

e w

ith

DA

PI

Brd

U

d

Control

siRNA

DONSON

siRNA

Denatured with 2M HCl

Merg

e w

ith

DA

PI

Brd

U

P < 0.001

P < 0.001

P < 0.001

P < 0.001


0

2

4

6

8

10

12

14

16

Perc

enta

ge o

f m

eta

phases

siRNA: Control DONSON Control DONSON Control DONSON

MMC HU

a

d

0

0.2

0.4

0.6

0.8

1

Untr MMC HU

Control siRNA

DONSON siRNA

Avera

ge r

adia

l chro

mosom

es / c

hro

mosom

e

exchanges p

er

meta

phases

c

Highly Fragmented

Pulverised

0

1

2

3

4

5

6

7

Untr MMC HU

Avera

ge c

hro

matid

gaps/b

reaks p

er

meta

phase

Control siRNA

DONSON siRNA

Untreated MMC HU

Untreated MMC HU

b

0

5

10

15

20

25

30

35

40

Untreated MMC HU

Control siRNA

DONSON siRNA

Perc

enta

ge o

f cells

with m

icro

nucle

i

Supplementary Figure 16: Replication stress exacerbates genome instability in DONSON

deficient cells.

Exposure of cells lacking DONSON to HU or MMC exacerbates micronuclei formation and

chromosome breakage. (a) Micronuclei formation in HeLa cells transfected with either control or

DONSON siRNA following exposure to either 4mM HU for 6 h, followed by 24 h recovery, or

50ng/ml MMC for 24 h was assessed using fluorescence microscopy (n = 3 independent

experiments). (b) Quantification of the chromatid gaps/breaks in metaphases from control or

DONSON siRNA transfected HeLa cells following exposure to HU or MMC as in (a). At least 50

metaphases per experiment were counted (n = 3 independent experiments). (c) Quantification of

chromosome exchanges and radial chromosomes in metaphases from control or DONSON siRNA

transfected HeLa cells following exposure to HU or MMC as in (a). At least 50 metaphases per

experiment were counted (n = 3 independent experiments). (d) Quantification of metaphases from

control or DONSON siRNA transfected HeLa cells, containing highly fragmented or pulverised

metaphases following exposure to HU or MMC as in (a) (n = 3 independent experiments). P values

were determined by a ’ t-test (a,b,c), or a chi-squared test.

P = 0.00052

P = 0.038

P = 0.098

P = 0.01

P = 0.031

P = 0.095

P < 0.001

P < 0.001

P < 0.001

P = 1.8E-08

P = 0.00063 P = 2.23E-05


0

5

10

15

20

25

30

35

40

Perc

enta

ge

of C

ldU

la

belle

d f

ork

s

WT1WT2AJ602AJ1794AJ1795AJ1796AJ1884

Stalled forks

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Untreated

Avera

ge n

um

ber

of

chro

mosom

al

aberr

atio

ns p

er

meta

phase

WT1

WT2

AJ602

AJ1794

AJ1795

AJ1796

AJ1884

WT1

WT2

P6

P12

P2

P9

P10-2

a

WT1

WT2

P6

P12

P2

P9

P10-2

0.0

5.0

10.0

15.0

20.0

25.0

Untreated

Perc

enta

ge o

f cells

with 5

3B

P1 a

nd

γ-H

2A

X f

oci

WT1

WT2

AJ602

AJ1794

AJ1795

AJ1796

AJ1884

WT1

WT2

P6

P12

P2

P9

P10-2

b

20 min 20 min

CldU IdU Harvest

Untreated

HU treated 20 min 20 min

Harvest

2 mM HU

2 h

CldU IdU

Supplementary Figure 17: Cells from patients with hypomorphic DONSON mutations exhibit genome

instability and replication fork abnormalities that are exacerbated by replication stress.

(a) Mutation of DONSON results in replication fork instability that is exacerbated by replication stress. Patient

derived fibroblasts were pulsed for 20 min with CldU, exposed to 2 mM HU for 2 h, and then pulsed with IdU for

20 min. DNA fibres were quantified, and the percentage of ongoing forks, new origin firing and stalled forks are

displayed (n = 3 independent experiments). Bottom right: Schematic of DNA fibre analysis. (b) DONSON patient

derived fibroblasts exhibit elevated levels of spontaneous 53BP1 and γ 2AX foci. Patient derived fibroblasts

were immunostained with antibodies to 53BP1 and γ 2AX, and the percentage of cells with 53BP1/γ 2AX foci

were quantified (n = 3 independent experiments). (c) Cells from DONSON patients exhibit spontaneous

chromosomal instability. Chromosomal aberrations in patient derived fibroblasts were quantified (n = 3

independent experiments). Throughout, P values were determined by a ’ t-test (a,b,c).

c

0

10

20

30

40

50

60

70

80

90P

erc

enta

ge o

f C

ldU

la

belle

d f

ork

s

Ongoing forks

0

2

4

6

8

10

12

Perc

enta

ge o

f C

ldU

la

belle

d f

ork

s

New origins + HU + HU

+ HU


0

2

4

6

WT1 P2 P9 WT2 P6 P12 P2

Left

fork

le

ngth

/rig

ht

fork

le

ngth

(ra

tio

)

P10-2 WT1 P9 P10-2 WT2 P6 P12

HU

Supplementary Figure 18: Cells from patients with hypomorphic DONSON mutations exhibit

increased replication fork instability

Quantification of DNA replication fork asymmetry in DONSON mutant patient-derived fibroblasts.

The plot indicates the ratio of left/right fork track lengths of bidirectional replication forks. The red

lines denote the median ratio (n = 3 independent experiments). Bottom: Schematic of DNA fibre

analysis.

20 min 20 min

CldU IdU Harvest

Untreated HU treated 20 min 20 min

Harvest

2 mM HU

2 h

CldU IdU


0

10

20

30

40

50

60

70

80

90

Perc

enta

ge

of C

ldU

la

belle

d f

ork

s

GFP-Donson WT + Con siRNA

GFP-Donson WT + Donson siRNA

GFP-Donson S28R + Donson siRNA

GFP-Donson M446T + Donson siRNA

GFP-Donson K489T + Donson siRNA

GFP-Donson WT + Donson siRNA + DOX

GFP-Donson S28R + Donson siRNA + DOX

GFP-Donson M446T + Donson siRNA +DOXGFP-Donson K489T + Donson siRNA +DOX

+ DOX

0

5

10

15

20

25

30

Perc

enta

ge o

f C

ldU

la

belle

d f

ork

s

GFP-DONSON WT + Control siRNA

GFP-DONSON WT + DONSON siRNA

GFP-DONSON S28R + DONSON siRNA

GFP-DONSON M446T+ DONSON siRNA

GFP-DONSON K489T+ DONSON siRNA

+ DOX

20 min 20 min

CldU IdU Harvest

+/- DOX

24 h

Stalled forks Ongoing forks

Supplementary Figure 19: The patient-derived M446T and K489T mutations fail to correct the

replication fork instability associated with DONSON depletion.

HeLa Flp-In/T-Rex cells from (Fig 2d) expressing GFP-tagged WT or mutant DONSON under the

control of a doxycycline-inducible promoter were transfected with DONSON siRNA. 48 h post-

transfection, 1 μ /ml doxycycline was added for 24 h. The cells were pulsed with CldU for 20 min,

followed by IdU for 20 min. DNA fibres were quantified, and the percentage of ongoing forks and

stalled forks are displayed (n = 2 independent experiments). ‘+ X’ indicates the addition of

doxycycline 24 h prior to incubation with the thymidine analogues. Bottom: Schematic of DNA fibre

analysis. P values were determined by a ’ t-test.

P = 0.01

P = 0.0002

P = 0.28

P = 0.39

P = 0.0007

P = 0.0003

P = 0.42

P = 0.10


0

5

10

15

20

25

30

35

40

Perc

enta

ge

of C

ldU

-la

belle

d fork

s

0

2

4

6

8

10

12

Perc

enta

ge

of C

ldU

-la

belle

d fork

s

Supplementary Figure 20: Re-expression of

WT DONSON in patient derived fibroblasts

restores replication fork stability.

DNA fibre analyses in hTERT-immortalised

DONSON patient fibroblasts complemented with

a retrovirus either containing human WT

DONSON, or an empty vector from (Fig 7).

Complemented fibroblasts were pulsed with

CldU, exposed to 2 mM HU for 2 h where

indicated, and then pulsed with IdU. The

percentage of ongoing replication forks, new

origin firing and stalled forks were quantified (n =

3 independent experiments). Quantification of

new origin firing and stalled forks presented in

(Fig 7d) have been included here for

comparison. Right: Schematic of DNA fibre

analysis. P values were determined by a

’ t-test.

New origins

Stalled forks

20 min 20 min

CldU IdU Harvest

Untreated


Harvest

2 mM HU

2 h

CldU IdU

0

10

20

30

40

50

60

70

80

90

Perc

enta

ge

of C

ldU

-la

belle

d fork

s

WT2

AJ602

AJ1795

AJ1796

AJ602 + Donson

AJ1795 + Donson

AJ1796 + Donson

WT2 + HU

AJ602 + HU

AJ1795 + HU

AJ1796 + HU

AJ602 + HU + Donson

AJ1795 + HU + Donson

AJ1796 + HU + Donson

WT2

P6

P2

P9

P6 + DONSON

P2 + DONSON

P9 + DONSON

Ongoing forks

P = 0.014

P = 0.021

P = 0.020

P = 0.008

P = 0.014

P = 0.002

P = 0.78

P = 0.75

P = 0.82

P = 0.011

P = 0.010

P = 0.039

P = 0.003

P = 0.017

P = 0.002

P = 0.005

P = 0.015

P = 0.002

+ HU

+ HU

+ HU


0

5

10

15

20

25

30

35

40

Perc

enta

ge o

f C

ldU

la

belle

d f

ork

s

+ATRi

0

10

20

30

40

50

60

70

80

90

AJ1795

AJ1795 + Donson

AJ1795 + HU

AJ1795 + Donson + HU

AJ1795 + ATRi

AJ1795 + Donson + ATRi

AJ1795 + HU + ATRi

AJ1795 + Donson + HU + ATRi

+ATRi

Perc

enta

ge o

f C

ldU

la

belle

d f

ork

s

P2

P2 + DONSON

P2

P2 + DONSON

P2

P2 + DONSON

P2 + DONSON

P2

0

2

4

6

8

10

12

Perc

enta

ge o

f C

ldU

la

belle

d f

ork

s

+ATRi

+ATRi

+HU

+HU

20 min 20 min

CldU IdU Harvest

Untreated


Harvest

2 mM HU

2 h

CldU IdU

HU + ATRi 20 min 20 min

Harvest

2 mM HU + 5 µM ATRi

2 h

CldU IdU

20 min 20 min

CldU IdU Harvest

+ ATRi

5 µM ATRi

Supplementary Figure 21: The replication

abnormalities exhibited by DONSON mutant patient

cells are epistatic with ATR inhibition.

Replication fork analysis of fibroblasts derived from

DONSON patient P2 expressing either empty vector or

WT DONSON in the presence or absence of HU and/or

ATRi. Cells were pulsed with CldU, exposed to 2 mM

HU, +/- 5μ ATR inhibitor for 2 h, and then pulsed with

IdU. DNA fibres were quantified, and the percentage of

ongoing forks, new origin firing and stalled forks are

displayed (n = 2 independent experiments). Right:

Schematic of DNA fibre analysis. P values were

determined by a ’ t-test.

New origins

Stalled forks

Ongoing forks

P = 0.004

P = 0.034

P = 0.88

P = 0.22

P = 0.99

P = 0.029 P = 0.21

P = 0.45

P = 0.001

P = 0.002

P = 0.42

P = 0.006


Supplementary Figure 22: Model depicting how DONSON mutations may lead to genome instability

and microcephaly.

DONSON is a component of the replisome, a very large macromolecular complex of over two hundred

proteins, including key replication machinery, the CMG complex (CDC45, MCM2-7 and GINS), PCNA, and

the replicative polymerases (Pol). (a) Upon replication stress (red diamond) and replication fork stalling, the

presence of functional DONSON ensures maintenance of fork stability. The ATR-dependent replication

stress response also contributes to ensuring replication fork stability, and promotes activation of the intra-S

and G2/M phase checkpoints, preventing transmission of DNA damage through to mitosis, and ensuring

genome stability. (b) In the absence of functional DONSON, stalled replication forks are cleaved by

structure-specific nucleases, such as MUS81 and XPF, leading to the generation of DSBs in S-phase.

Impaired S-phase and G2/M checkpoint function in the absence of DONSON facilitates the transit of DNA

damage into subsequent phases of the cell cycle, resulting in elevated mitotic chromosome breaks that will

increase cell death. In patients with mutations in DONSON, such increased cell death would lead to reduced

numbers of cells being generated during development, resulting in the microcephaly and growth failure

observed.


Supplementary Table 2. Common ancestral haplotype (blue) flanking the DONSON locus in families P1, P2, P3 and P6.

SNP positionb gene coding Ref Alt P1-1 P1-2 P2 P3 P6 MAFc

rs121913215 34804930 IFNGR2 intronic A T AT AT AT AT AA 0.0010

rs75398504 34839530 TMEM50B intronic G A GA GA GA GA GG 0.0006

rs144266803 34861444 DNAJC28 T86I G A GA GA GA GA GA 0.0010

rs148155071 34876289 GART 3ʹUTR A T AT AT AT AT AT 0.0026

rs151206034 34889888 GART M577T A G AG AG AG AG AG 0.0010

rs139633075 34897281 GART L365V G C GC GC GC GC GC 0.0010

rs187476997 34902228 GART intronic T C TC TC TC TC TC 0.0016

rs142038738 34903861 GART E177D C A CA CA CA CA CA 0.0009

rs148794591 34924549 SON A1004A T C TC TC TC TC TC 0.0010

rs142751481 34925936 SON I1467V A G AG AG AG AG AG 0.0010

rs146664036 34951753 DONSON K489T T G TG TG TG TG TG 0.0010

rs192585552 34956005 DONSON c.786-33 T C TC TC TC TC TC 0.0005

no rsIDa 34960866 DONSON S28R T G TG TG TG TG TG -

rs190141427 34967690 CRYZL1 3ʹUTR T G TG TG TG TG TG 0.0006

rs184229251 34989145 CRYZL1 intronic T C TC TC TC TC TC 0.0006

rs570210988 36261887 RUNX1 intronic C C CC CC CT CC CC 0.0002

Analysis of rare variants identified through exome sequencing demonstrated a common ancestral haplotype extending 127.7 kb in DONSON patients heterozygous for the ‘haplotype’ disease allele. Parents of P2 and P3 were also exome sequenced, allowing phasing of alleles across this haplotype. No other variants were identified in trans in the genes within this region in any individuals apart from DONSON – the in trans variants in DONSON are omitted here for clarity. aThis variant lies within a GC rich region and therefore is not covered in many control population datasets. bGenomic position using hg19 coordinates. cMinor allele frequencies taken from ExAC or 1000 Genomes data.


Supplementary Note:

Fluorescence Cross-Correlation Spectroscopy (FCCS)

Fluorescence imaging and fluorescence (cross)-correlation

spectroscopy (FCCS) measurements were performed on a uniquely modified

confocal laser scanning microscopy system, the ConfoCor3 (Carl Zeiss, Jena,

Germany), consisting of the Zeiss LSM780 inverted setup and comprising

Diode 405 nm, Ar multiline 458, 488 and 514 nm, DPSS 561 nm and HeNe

633 nm lasers. It enables detection using silicon Avalanche Photo Detectors

(APDs) (SPCM-AQR-1X; PerkinElmer, USA) for imaging and FCS. Images

were recorded at a 1024X1024 pixel resolution. The C-Apochromat 40x/1.2 W

UV-VIS-IR objective was used throughout. Fluorescence intensity fluctuations

were recorded in arrays of 10 consecutive measurements, each measurement

lasting 10 s. Averaged curves were analyzed using the ZEN software for

online data analysis or exported and fitted offline. In either case, the nonlinear

least square fitting of the autocorrelation curve was performed using the

Levenberg–Marquardt algorithm. Quality of the fitting was evaluated by visual

inspection and by residuals analysis. Control FCS measurements to assess

the detection volume were routinely performed prior to data acquisition, using

dilute solutions (10 𝑛𝑀 ) of Alexa488 and CFTM568 dyes. Highly purified

double-stranded DNA, in which each strand carries one fluorophore of

Alexa488 and Atto565, was used as a cross-correlation standard. 85% cross-

correlation was measured using the FCCS standard, whereas 13% cross-

correlation (due to cross-talk) was measured with the two dyes in solution

(data not shown). The variation between independent measurements reflects

variations between cells, rather than imprecision of FCS measurements.


FCS/FCCS measurements of GFP-DONSON/RFP-PCNA and GFP/RFP-

PCNA expressing cells

HeLa cells stably expressing GFP-DONSON and mCherry-PCNA (construct

kindly provided by C. Lukas, Copenhagen; referred to as RFP-PCNA) were

grown under standard conditions overnight on chambered coverslips (μ-slide,

8 well, Ibidi) and the growth medium was replaced by L-15 medium (Leibovitz)

(Sigma) immediately prior to FCS/FCCS measurements. Cells expressing low

levels of RFP-PCNA/GFP-DONSON or RFP-PCNA/GFP were used for

FCS/FCCS measurements. Measurements were made in weakly expressing

cells only. As the concentration of RFP-PCNA in replication foci was observed

to be several-fold higher than that of DONSON-GFP and GFP, only cells with

similar GFP/RFP ratios were used for cross-correlation analysis and

comparable GFP-DONSON and GFP only concentrations used for

comparison.

Background on Fluorescence Microscopy Imaging and FCS

As described above, an individually modified instrument (Zeiss, LSM780,

ConfoCor 3) with fully integrated FCS/CLSM optical pathways was used for

imaging. The detection efficiency of CLSM imaging was significantly improved

by the introduction of APD detectors. As compared to PMTs, which are

normally used as detectors in conventional CLSM, the APDs are

characterized by higher quantum yield and collection efficiency – about 70 %

in APDs as compared to 15 – 25 % in PMTs, higher gain, negligible dark


current and better efficiency in the red part of the spectrum. Enhanced

fluorescence detection efficiency enabled image collection using fast scanning

(1 − 5 µ𝑠/𝑝𝑖𝑥𝑒𝑙). This enhances further the signal-to-noise-ratio by avoiding

fluorescence loss due to triplet state formation, enabling fluorescence imaging

with single-molecule sensitivity. In addition, low laser intensities

(150– 750 µ𝑊) could be applied for imaging, significantly reducing the photo-

toxicity.

FCS/FCCS measurements are performed by recording fluorescence

intensity fluctuations in a very small observation volume element (OVE) of a

prolate ellipsoid shape (about 200𝑛𝑚 wide and about 1µ𝑚 long) that is placed

in GFP-DONSON/RFP-PCNA or control GFP/RFP-PCNA HeLa cells by

focusing the laser light through the microscope objective and by collecting the

fluorescence light through the same objective using a pinhole in front of the

detector to block out-of-focus light. Boxed areas of 0.7𝜇𝑚2 in Fig. 4e indicate

z-projections of larger representative regions in which the Observation

Volume Element (OVE) of:

𝑉𝑂𝑉𝐸 = 𝜋32 ∙ 𝑤𝑥𝑦

2 ∙ 𝑧0 = 5.57 ∙ 0.1847𝟐 ∙ 1 = 0.223 ∙ 10−18 𝑚3 = 0.19 ∙ 10−15𝐿

was placed for FCCS measurements. We established that observed

fluorescence intensity fluctuations are caused by fluorescently labeled GFP-

DONSON or RFP-PCNA molecules passing through the OVE and that the

characteristic decay times of the autocorrelation functions do not depend on

the concentration of GFP-DONSON or RFP-PCNA (Supplementary Fig. 13).

Therefore, we analyzed the diffusion of the two proteins in the nucleus, using

temporal autocorrelation analysis.


In temporal autocorrelation analysis we first derive the autocorrelation

function G():

𝐺(𝜏) = 1 +⟨𝛿𝐼(𝑡)∙𝛿𝐼(𝑡+𝜏)⟩

⟨𝛪(𝑡)⟩2 ,

where 𝛿𝐼(𝑡) = 𝐼(𝑡)– ⟨𝛪(𝑡)⟩ is the deviation from the mean intensity at time t

and 𝛿𝐼(𝑡 + 𝜏) = 𝐼(𝑡 + 𝜏)– ⟨𝛪(𝑡)⟩ is the deviation from the mean intensity at

time 𝑡 + 𝜏. For further analysis, an autocorrelation curve (ACC) is derived by

plotting 𝐺(𝜏) as a function of the lag time, i.e. the autocorrelation time .

To derive information about molecular numbers and their

corresponding diffusion time, the experimentally obtained ACCs are

compared to autocorrelation functions derived for different model systems. A

model describing free three-dimensional diffusion of two components was

used for fitting Cross-Correlation Curves (CCCs) in this study, whereas a two-

component model with triplet correction was used for fitting ACCs:

𝐺(𝜏) = 1 +1

𝑁

(

1−𝑦

(1+𝜏

𝜏𝐷1)∙√1+

𝑤𝑥𝑦2 𝜏

𝑤𝑧2𝜏𝐷1

+𝑦

(1+𝜏

𝜏𝐷2)∙√1+

𝑤𝑥𝑦2 𝜏

𝑤𝑧2𝜏𝐷2)

Te

T

T

11

In the above equation, N is the average number of molecules in the

OVE; y is the fraction of the slowly moving GFP-DONSON and RFP-PCNA

molecules; 𝜏𝐷1 is the diffusion time of the free GFP-DONSON and RFP-PCNA

molecules; 𝜏𝐷2 is the diffusion time of GFP-DONSON and RFP-PCNA

molecules undergoing interactions; 𝑤𝑥𝑦 and 𝑤𝑧 are radial and axial

parameters, respectively, related to spatial properties of the OVE; T is the

average equilibrium fraction of molecules in the triplet state; and T the triplet

correlation time related to rate constants for intersystem crossing and the


triplet decay. Spatial properties of the detection volume, represented by the

square of the ratio of the axial and radial parameters ((𝑤𝑧

𝑤𝑥𝑦)2

), are determined

in calibration measurements performed using a 10 𝑛𝑀 solution of Alexa488

and CFTM568 for which the diffusion coefficient (D) is known. The diffusion

time, 𝜏𝐷, measured by FCS, is related to the translation diffusion coefficient D

by:

𝜏𝐷 =𝑤𝑥𝑦2

4𝐷.


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